1. Field of the Invention
The present invention relates to a method for identifying an olfactory receptor included in one olfactory cell.
2. Background of the Related Art
An olfactory receptor is a trimeric G protein-coupled receptor (hereinafter, referred to as “GPCR”). More particularly, an olfactory receptor is one kind of trimeric G protein-coupled seven-transmembrane receptors.
FIG. 4 shows a mechanism that a stimulus of an odor molecule to a cell membrane is converted into an electric signal.
The olfactory receptor is a membrane protein which is expressed in a cell membrane. The cell membrane is mainly composed of a lipid bilayer membrane. A lipid bilayer membrane has a structure of two layers each consisting of phospholipid molecules lined with high density. This lipid bilayer membrane is schematically shown in the center of FIG. 4. In FIG. 4, the outside of the cell is above the upper part of the lipid bilayer membrane. On the other hand, the inside of the cell is below the lower part of the lipid bilayer membrane. The trimeric G protein is placed in the vicinity of the olfactory receptor.
The trimeric G protein is a heterotrimer is composed of an alpha subunit (Gαolf), a beta-subunit (Gβ), and a gamma subunit (Gγ). The cell contains adenylate cyclase. In FIG. 4, the adenylate cyclase is referred to as “AC”. To be more exact, the adenylate cyclase is a transmembrane-type protein. A protein RTP1S has a function to assist the olfactory receptors to be expressed in the cell membrane. It is noted that the protein RTP1S is not directly associated with the mechanism.
Next, the mechanism is described. The odor molecule binds to the olfactory receptor. The binding leads to separation of the trimeric G protein into the alpha subunit (Gαolf) and a beta-gamma complex. The beta-gamma complex consists of the subunit Gβ and the subunit γ. The separated Gαolf activates the adenylate cyclase (AC). The activated adenylate cyclase (AC) converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP).
The cyclic adenosine monophosphate (cAMP) activates an ion channel, more particularly, for example, a cyclic nucleotide gated ion channel (CNG). The activation allows an ion to be transported from the inside of the cell to the outside of the cell, or from the outside of the cell to the inside of the cell. The degree of the transport of the ion can be measured as an electric signal.
The mouse contains approximately 1,300 kinds of the olfactory receptors. The olfactory receptors are collectively referred to as “Olfr_n”. Generally, n represents a natural number. For example, an olfactory receptor having a name of “Olfr943” is known.
One olfactory cell includes one kind of the olfactory receptor. One olfactory cell does not include two or more kinds of the olfactory receptors.
When an olfactory receptor is not expressed in a cell transfected with a gene sequence coding for an olfactory receptor, a skilled person who has acquired one olfactory cell is not able to identify the olfactory receptor included in the one olfactory cell.
In order to solve this problem, Non Patent Literature 1 discloses a method for identifying an olfactory receptor included in one olfactory cell among Olfr480, Olfr544, Olfr545, Olfr586, Olfr642, Olfr661, Olfr672, Olfr690, Olfr744, and Olfr749, using a pair of primers represented by SEQ ID: 03 and SEQ ID: 04, a pair of primers represented by SEQ ID: 03 and SEQ ID: 05, or a pair of primers represented by SEQ ID: 03 and SEQ ID: 06.
Non Patent Literature 2 discloses a method for determining whether or not an olfactory receptor included in one olfactory cell is Olfr16, using a pair of primers represented by SEQ ID: 01 and SEQ ID: 07.
Non Patent Literature 3 discloses a method for identifying an olfactory receptor included in one olfactory cell among Olfr1056, Olfr1366, and Olfr1484, using a pair of primers represented by SEQ ID: 03 and SEQ ID: 08.